The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is an important pest of cotton that also feeds on other crops and many wild hosts. In the mid-South, where tarnished plant bugs in cotton are controlled exclusively with synthetic insecticides, infestations resulted in >29% of all yield loss attributed to insect damage and the control costs exceeded US$10 per acre in 2013. They have developed resistance to the most commonly used insecticides. Estimations of gene flow and migration are important to understand the spread of resistance in tarnished plant bug populations. Here, we analyzed tarnished plant bugs collected from July to September, 2006, to estimate population genetic parameters using 13 microsatellite markers. Our data indicated that tarnished plant bug populations in the study area had undergone a population bottleneck and all loci deviated from Hardy—Weinberg equilibrium in one or more collections. Bayesian simulations and factorial correspondence analysis indicated the presence of two genetic clusters in the tarnished plant bug populations in the study area. Proportions of insects separated into the two genetic clusters changed from July to September. Genetic differentiation and reduced gene flow between populations were also observed. We postulate that while random genetic drift and gene flow may have contributed to the seasonal variations observed in the study populations, selection by insecticide sprays in cotton during 2005–2006 as well as other extrinsic factors could also have played a significant role in the temporal variation in genetic structure observed in the tarnished plant bug populations.